This study aimed to assess how the wear that brushing promotes affects laser-irradiated enamel microhardness after cariogenic challenge in vitro. Forty fragments measuring were randomly assigned to four groups according to the enamel surface treatment: G1—control, G2—-laser irradiation, G3—brushing, and G4— laser irradiation + brushing. A laser device emitting at 10.6 μm was used (, energy per , and ). Specimens belonging to groups G3 and G4 were brushed (80,000 strokes) with a brushing simulator using toothpaste. Next, the samples were challenged with acid: the specimens were immersed in demineralizing and remineralizing solutions for 8 days. The acid resistance of enamel was evaluated by cross-sectional microhardness tests. The area under the curve () was calculated. Analysis of variance (ANOVA) one-away and Fisher’s test were performed for the statistical analysis (). Group G2 specimens () were statistically different from specimens belonging to groups G1 (), G3 (), and G4 (), which were statistically similar. The brushing time used in the present study probably wore the -lased enamel, so demineralization could not be prevented in the brushed group.